Persistent infection with Helicobacter pylori is a significant risk factor for a number of gastric disorders in humans, including peptic ulceration and gastric adenocarcinomas. The vacuolating cytotoxin (VacA) is an important virulence factor that mounting evidence indicates is involved in multiple aspects of H. pylori-mediated colonization and persistence. The broad objective of this research program is to identify how the cellular modulating activities of VacA contribute to the pathogenesis strategies of H. pylori within the host. In this application, we propose to explore two important aspects of VacA cellular intoxication. In Specific Aim 1, we propose experiments to investigate the mechanism underlying recent observations that VacA induces caspase-dependent programmed cell death in a mitochondrial-dependent manner. In Specific Aim 1, we will explore the overall hypothesis that VacA localizes to mitochondria and elaborates a biochemical activity that modulates mitochondrial membrane permeability. We will test a number of predictions of this hypothesis related to the mechanism of VacA-induced changes in membrane permeability, and how these changes relate to downstream consequences within the cell. Moreover, we will test the prediction that VacA is "hard-wired" with sequences or motifs to target the toxin to mitochondria and then engage existing mitochondrial import machinery. In these studies, we will employ both cultured cell lines, as well as isolated mitochondria. The proposed experiments are important for revealing aspects of an emerging strategy used by several pathogens to directly target mitochondria as a strategy for modulating host cell death. In Specific Aim 2, we will explore the interactions of VacA with specialized microdomains of the plasma membrane of target cells that have been demonstrated to be important for cellular activity. We will explore the overall hypothesis that VacA associates with and functionally lipid rafts on the plasma membrane of target host cells. In this Aim, we will investigate the dependency of different VacA cellular activities on lipid rafts, as well as the functional relationship between lipid rafts and a known receptor for VacA. We will then explore how the lipid composition affects VacA association with rafts. Finally, we will identify and characterize VacA sequences and/or motifs that promote binding to lipid rafts. These studies will reveal novel aspects of the mechanisms underlying cellular intoxication, as well as new approaches for blocking cellular intoxication. Because it is estimated that the prevalence of H. pylori infection in developed countries is 20-50%, and 70-90% in developing countries, the importance of developing efficacious vaccines, chemotherapeutics, and diagnostics for H. pylori cannot be overstated. These studies will not only contribute to our understanding of the fundamental mechanisms of VacA-mediated cytotoxicity, but may reveal novel strategies for blocking or minimizing the consequences of VacA-mediated cellular intoxication during infection.